U.S. patent application number 10/491263 was filed with the patent office on 2005-01-27 for 4[piperidin-4-yliden-(3-carbamoylphenyl)methyl]benzamide derivatives and their use for the treatment of pain spinal injuries or gastrointestinal disorders.
Invention is credited to Brown, William, Walpole, Christopher, Wei, Zhongyong.
Application Number | 20050020629 10/491263 |
Document ID | / |
Family ID | 20285553 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020629 |
Kind Code |
A1 |
Brown, William ; et
al. |
January 27, 2005 |
4[piperidin-4-yliden-(3-carbamoylphenyl)methyl]benzamide
derivatives and their use for the treatment of pain spinal injuries
or gastrointestinal disorders
Abstract
Compounds of general formula (I) wherein R.sup.1 is selected
from any one of phenyl, pyridinyl, thienyl, furanyl, imidazolyl,
pyrrolyl and triazolyl; where each R.sup.1 phenyl ring and R.sup.1
heteroaromatic ring may optionally and independently be further
substituted by 1, 2 or 3 substituents selected from straight and
branched C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3, C.sub.1-C.sub.6
alkoxy, chloro, fluoro, bromo, and iodo. The substitutions on
thephenyl ring and on the heteroaromatic ring may take place in any
position on said ring systems; are disclosed and claimed in the
present application, as well as their pharmaceutically acceptable
salts and pharmaceutical compositions comprising the novel
compounds and their use in therapy, inparticular in the management
of pain.
Inventors: |
Brown, William; (Quebec,
CA) ; Walpole, Christopher; (Quebec, CA) ;
Wei, Zhongyong; (Quebec, CA) |
Correspondence
Address: |
ASTRA ZENECA PHARMACEUTICALS LP
GLOBAL INTELLECTUAL PROPERTY
1800 CONCORD PIKE
WILMINGTON
DE
19850-5437
US
|
Family ID: |
20285553 |
Appl. No.: |
10/491263 |
Filed: |
September 2, 2004 |
PCT Filed: |
October 2, 2002 |
PCT NO: |
PCT/SE02/01804 |
Current U.S.
Class: |
514/318 ;
514/317; 514/326; 546/193; 546/207; 546/210; 546/212; 546/233 |
Current CPC
Class: |
C07D 401/06 20130101;
A61P 1/00 20180101; A61P 25/00 20180101; C07D 417/06 20130101; A61P
25/04 20180101; C07D 211/70 20130101; A61P 25/02 20180101; C07D
409/06 20130101; C07D 405/06 20130101 |
Class at
Publication: |
514/318 ;
514/326; 514/317; 546/193; 546/207; 546/210; 546/212; 546/233 |
International
Class: |
A61K 031/4545; A61K
031/454; C07D 49/02; C07D 45/02; C07D 43/02; C07D 41/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2001 |
SE |
0103313-3 |
Claims
1. A compound of the formula I 22wherein R.sup.1 is selected from
any one of (i) phenyl 23(ii) pyridinyl 24(iii) thienyl 25(iv)
furanyl 26(v) imidazolyl 27(vi) triazolyl 28(vii) pyrrolyl 29and
(viii) thiazolyl 30where each R.sup.1 phenyl ring and R.sup.1
heteroaromatic ring may independently be further substituted by 1,
2 or 3 substituents independently selected from straight and
branched C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3, C.sub.1-C.sub.6
alkoxy, chloro, fluoro, bromo, and iodo; as well as salts
thereof.
2. A compound according to claim 1 wherein R.sup.1 is selected from
any one of (i) phenyl 31(ii) pyridinyl 32(iii) thienyl 33and (iv)
furanyl 34
3. A compound according to claim 1, wherein each R.sup.1 phenyl
ring and R.sup.1 heteroaromatic ring may optionally and
independently be further substituted by 1, 2 or 3 substituents
independently selected from methyl, CF.sub.3, chloro, fluoro,
bromo, and iodo.
4. A compound according to claim 1, selected from any one of
N,N-Diethyl-4-[piperidin-4-ylidene(3-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(2-thiophene)methyl-piperidin-4-ylidene-(3-carbamoylphen-
yl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(2-furfuryl-piperidin-4-ylidene-(3-
-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(3-furfuryl-piperidi-
n-4-ylidene-(3-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(2-pyr-
idine)methyl-piperidin-4-ylidene-(3-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(3-thiophene)methyl-piperidin-4-ylidene-(3-carbamoylphen-
yl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(2-thiazole)methyl-piperidin-4-yli-
dene-(3-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(3-pyridine)m-
ethyl-piperidin-4-ylidene-(3-carbamoylphenyl)-methyl]-benzamide;
N,N-Diethyl-4-[1-(2-pyrrole)methyl-piperidin-4-ylidene-(3-carbamoylphenyl-
)-methyl]-benzamide;
N,N-Diethyl-4-[1-(4-pyridine)methyl-piperidin-4-ylide-
ne-(3-carbamoylphenyl)-methyl]-benzamide; and
N,N-Diethyl-4-[1-(4-pyridine-
)methyl-piperidin-4-ylidene-(3-carbamoylphenyl)-methyl]-benzamide.
5. A compound according to claim 1, in form of its hydrochloride,
dihydrochloride, sulfate, tartrate, ditrifluoroacetate or citrate
salts.
6. A compound according to any one of claims 1-5 for use in
therapy.
7. A compound according to claim 6, wherein the therapy is pain
management.
8. A compound according to claim 6, wherein the therapy is directed
towards gastrointestinal disorders.
9. A compound according to claim 6, wherein the therapy is directed
towards spinal injuries.
10. A compound according to claim 6, wherein the therapy is
directed to disorders of the sympathetic nervous system.
11. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of pain.
12. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of
gastrointestinal disorders.
13. Use of a compound according to formula I of claim 1 for the
manufacture of a medicament for use in the treatment of spinal
injuries.
14. A pharmaceutical composition comprising a compound of the
formula I according to claim 1 as an active ingredient, together
with a pharmaceutically acceptable carrier.
15. A method for the treatment of pain, whereby an effective amount
of a compound of the formula I according to claim 1 is administered
to a subject in need of pain management
16. A method for the treatment of gastrointestinal disorders,
whereby an effective amount of a compound of the formula I
according to claim 1, is administered to a subject suffering from
said gastrointestinal disorder.
17. A method for the treatment of spinal injuries, whereby an
effective amount of a compound of the formula I according to claim
1, is administered to a subject suffering from said spinal injury.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to novel compounds, to a
process for their preparation, their use and pharmaceutical
compositions comprising the novel compounds. The novel compounds
are useful in therapy, and in particular for the treatment of
pain.
BACKGROUND OF THE INVENTION
[0002] The .delta. receptor has been identified as having a role in
many bodily functions such as circulatory and pain systems. Ligands
for the .delta. receptor may therefore find potential use as
analgesics, and/or as antihypertensive agents. Ligands for the
.delta. receptor have also been shown to possess immunomodulatory
activities.
[0003] The identification of at least three different populations
of opioid receptors (.mu., .delta. and .kappa.) is now well
established and all three are apparent in both central and
peripheral nervous systems of many species including man. Analgesia
has been observed in various animal models when one or more of
these receptors has been activated.
[0004] With few exceptions, currently available selective opioid
.delta. ligands are peptidic in nature and are unsuitable for
administration by systemic routes. One example of a nonpeptidic
.delta.-agonist is SNC80 (Bilsky E. J. et al., Journal of
Pharmacology and Experimental Therapeutics, 273(1), pp. 359-366
(1995)). There is however still a need for selective
.delta.-agonists having not only improved selectivity, but also an
improved side-effect profile.
[0005] Thus, the problem underlying the present invention was to
find new analgesics having improved analgesic effects, but also
with an improved side-effect profile over current .mu. agonists, as
well as having improved systemic efficacy.
[0006] Analgesics that have been identified and exist in the prior
art have many disadvantages in that they suffer from poor
pharmacokinetics and are not analgesic when administered by
systemic routes. Also, it has been documented that preferred
.delta. agonist compounds, described within the prior art, show
significant convulsive effects when administered systemically.
[0007] We have now found that certain compounds not specifically
disclosed by, but included within the scope of WO 98/28275, exhibit
surprisingly improved .delta.-agonist properties and in vivo
potency.
OUTLINE OF THE INVENTION
[0008] The novel compounds according to the present invention are
defined by the formula I wherein 1
[0009] R.sup.1 is selected from any one of
[0010] (i) phenyl; 2
[0011] (ii) pyridinyl 3
[0012] (iii) thienyl 4
[0013] (iv) furanyl 5
[0014] (v) imidazolyl 6
[0015] (vi) triazolyl 7
[0016] (vii) pyrrolyl 8
[0017] and
[0018] (viii) thiazolyl 9
[0019] where each R.sup.1 phenyl ring and R.sup.1 heteroaromatic
ring may optionally and independently be further substituted by 1,
2 or 3 substituents independently selected from straight and
branched C.sub.1-C.sub.6 alkyl, NO.sub.2, CF.sub.3, C.sub.1-C.sub.6
alkoxy, chloro, fluoro, bromo, and iodo. The substitutions on the
phenyl ring and on the heteroaromatic ring may take place in any
position on said ring systems.
[0020] Particularly, novel compounds according to the present
invention are defined by the formula I
[0021] wherein R.sup.1 is selected from any one of
[0022] (i) phenyl 10
[0023] (ii) pyridinyl 11
[0024] (iii) thienyl 12
[0025] and
[0026] (iv) furanyl 13
[0027] Within the scope of the invention are also salts and
enantiomers of the compounds of the formula 1.
[0028] When the phenyl ring and the heteroaromatic ring(s) are
substituted, the preferred substituents are independently selected
from any one of CF.sub.3, methyl, iodo, bromo, fluoro and
chloro.
[0029] The novel compounds of the present invention are useful in
therapy, especially for the treatment of various pain conditions
such as chronic pain, neuropathic pain, acute pain, cancer pain,
pain caused by rheumatoid arthritis, migraine, visceral pain etc.
This list should however not be interpreted as exhaustive.
[0030] Compounds of the invention are useful as immunomodulators,
especially for autoimmune diseases, such as arthritis, for skin
grafts, organ transplants and similar surgical needs, for collagen
diseases, various allergies, for use as anti-tumour agents and anti
viral agents.
[0031] Compounds of the invention are useful in disease states
where degeneration or dysfunction of opioid receptors is present or
implicated in that paradigm. This may involve the use of
isotopically labelled versions of the compounds of the invention in
diagnostic techniques and imaging applications such as positron
emission tomography (PET).
[0032] Compounds of the invention are useful for the treatment of
diarrhoea, depression, anxiety, uninary incontinence, various
mental illnesses, cough, lung oedema, various gastro-intestinal
disorders, spinal injury and drug addiction, including the
treatment of alcohol, nicotine, opioid and other drug abuse and for
disorders of the sympathetic nervous system for example
hypertension.
[0033] Compounds of the invention are useful as an analgesic agent
for use during general anaesthesia and monitored anaesthesia care.
Combinations of agents with different properties are often used to
achieve a balance of effects needed to maintain the anaesthetic
state (e.g. amnesia, analgesia, muscle relaxation and sedation).
Included in this combination are inhaled anaesthetics, hypnotics,
anxiolytics, neuromuscular blockers and opioids.
[0034] Also within the scope of the invention is the use of an), of
the compounds according to the formula I above, for the manufacture
of a medicament for the treatment of any of the conditions
discussed above.
[0035] A further aspect of the invention is a method for the
treatment of a subject suffering from any of the conditions
discussed above, whereby an effective amount of a compound
according to the formula I above, is admininstered to a patient in
need of such treatment.
[0036] A further aspect of the present invention is intermediates
of the general formula II 14
[0037] wherein PG is a urethane protecting group such as Boc or
CBZ, or a benzyl or substituted benzyl protecting group, such as
2,4-dimethoxybenzyl.
[0038] Methods of Preparation
EXAMPLES
[0039] The invention will now be described in more detail by the
following Schemes and Examples, which are not to be construed as
limiting the invention. 15
[0040] Scheme 1: Synthetic Route to Compounds of the Present
Invention
[0041]
N,N-Diethyl-4-[N-Boc-piperidin-4-ylidene(3-carboxyphenyl)-methyl]-b-
enzamide (2a).
[0042] A mixture of
4-[bromo-(4-diethylcarbamoyl-phenyl)-methylene]-piperi-
dine-1-carboxylic acid tert-butyl ester (1a, 451 mg, 1.0 mmol),
3-carboxyphenyl boronic acid (330 mg, 2.0 mmol), 2M
Na.sub.2CO.sub.3 (3 mL), and tetrakis(triphenyl phosphine)
palladium(0) (25 mg) in toluene (degassed, 10 mL) and ethanol
(degassed, 10 mL) was refluxed at 90.degree. C. for 4 hrs under
N.sub.2. The reaction mixture was then quenched with aqueous
NH.sub.4Cl after cooling down to 0.degree. C., and extracted with
ethyl acetate (2.times.50 mL). The combined organic phases were
washed with brine, dried over MgSO.sub.4, and evaporated to give a
crude product, which was purified by flash silica gel column to
provide the desired compound 2a (345 mg, 70%): .sup.1H NMR
(CDCl.sub.3) .delta. 1.15 (3 H, br m, CH.sub.3CH.sub.2--), 1.23 (3
H, br m, CH.sub.3CH.sub.2--), 1.47 (9 H, s, C(CH.sub.3).sub.3),
2.31 (2 H, m, piperidine CH--), 2.35 (2 H, m, piperidine CH--),
3.30 (2 H, br m, CH.sub.3CH.sub.2N--), 3.48 (4 H, m, piperidine
CH--), 3.54 (2 H, br m, CH.sub.3CH.sub.2N--), 7.14 (2 H, d, J=8.0
Hz, ArH), 7.24 (1 H, m, ArH), 7.33 (2 H, d, J=8.0 Hz, ArH), 7.42 (1
H, t, J=7.6 Hz, ArH), 7.86(1 H, s, ArH), 7.97 (1 H, d, J=7.6 Hz,
ArH). IR (NaCl)2976, 1718, 1691, 1598, 1430, 1233, 1166
cm.sup.-1.
[0043]
N,N-Diethyl-4-[1-benzyl-piperidin-4-ylidene-3-carboxyphenyl)-methyl-
]-benzamide (2b).
[0044] Method as for 2a using 1b (441 mg, 1.0 mmol) and
3-carboxyphenyl boronic acid (330 mg, 2.0 mmol) provided 2b (325
mg, 67%): .sup.1H NMR (CDCl.sub.3) .delta. 1.11 (3 H, br m,
CH.sub.3CH.sub.2--), 1.22 (3 H, br m, CH.sub.3CH.sub.2--), 2.63 (4
H, m, piperidine CH--), 2.90 (4 H, m, piperidine CH--), 3.26 (2 H,
br m, CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--),
4.00 (2 H, s, CH.sub.2N--), 7.11 (2 H, d, J=8.0 Hz, ArH), 7.16 (1H,
m, ArH), 7.30 (6 H, m, ArH), 7.42 (2 H, m, ArH), 7.84 (1 H, s,
ArH), 7.96 (1 H, d, J=7.6 Hz, ArH).
[0045]
N,N-Diethyl-4-[1-benzylpiperidin-4-ylidene(3-carbamoylphenyl)-methy-
l]-benzamide (3b).
[0046] 241 mg (0.5 mmol) of
N,N-diethyl-4-[piperidin-4-ylidene(3-carboxyph-
enyl)-methyl]-benzamide (2b), 780 mg (1.5 mmol) of PyBOP, and 200
mg (1.5 mmol) of HOBt were dissolved in 4 mL DMF. 0.58 mL (4 mmol)
of DIPEA and 50 mg (10.0 mmol) of NH.sub.4Cl were added
successively. After stirred for 0:5 h at room temperature, the
reaction mixture was quenched with water and ethyl ether. The white
precipitates were collected as the desired product (3b, 152 mg,
63%): .sup.1H NMR (CDCl.sub.3) .delta. 1.10 (3 H, br m,
CH.sub.3CH.sub.2--), 1.21 (3 H, br m, CH.sub.3CH.sub.2--), 2.62 (4
H, m, piperidine CH--), 2.89 (4 H, m, piperidine CH--), 3.26 (2 H,
br m, CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--),
4.00 (2 H, s, CH.sub.2N--), 7.11 (2 H, d, J=8.0 Hz, ArH), 7.16 (1H,
m, ArH), 7.30(6 H, m, ArH), 7.42 (2 H, m, ArH), 7.84 (1 H, s, ArH),
7.96 (1 H, d, J=7.6 Hz, ArH); Anal.Calcd.for
C.sub.31H.sub.35N.sub.3O.sub.2 6.0 HCl: C, 53.16%; H, 5.90%; Found:
C, 53.07%; H, 5.54 %.
[0047]
N,N-Diethyl-4-[piperidin-4-ylidene(3-carbamoylphenyl)-methyl]-benza-
mide (4).
[0048] Method as for 3b using 2a (150 mg, 0.30 mmol) provided 3a
(105 mg, 70%): .sup.1H NMR (CDCl.sub.3) .delta. 1.14 (3 H, br m,
CH.sub.3CH.sub.2--), 1.22 (3 H, br m, CH.sub.3CH.sub.2--), 1.46 (9
H, s, C(CH3)3), 2.28 (2 H, m, piperidine CH--), 2.33 (2 H, m,
piperidine CH--), 3.30 (2 H, br m, CH.sub.3CH.sub.2N--), 3.46 (4 H,
m, piperidine CH--), 3.55 (2 H, br m, CH.sub.3CH.sub.2N--), 7.13 (2
H, d, J=8.0 Hz, ArH), 7.30 (3 H, m, ArH), 7.39(1 H, t, J=7.6 Hz,
ArH), 7.61 (1 H, s, ArH), 7.68 (1 H, d, J=7.6 Hz, ArH).
[0049] The above product (3a) was treated with 4.0 M HCl in dioxane
(10 mL) at room temperature for 4 h. After evaporation, the residue
was dissolved in H.sub.2O (10 mL) and impurities were extracted
with ethyl acetate (2.times.20 mL). The aqueous phase was basified
with NH.sub.4OH and extracted with ethyl acetate (3.times.20 mL).
The combined organic phases were washed with brine, dried over
MgSO.sub.4 and evaporated to give 4 in quantitative yield: .sup.1H
NMR (CDCl.sub.3) .delta. 1.15 (3 H, br m, CH.sub.3CH.sub.2--), 1.23
(3 H, br m, CH.sub.3CH.sub.2--), 1.90 (2 H, br, NH.sub.2), 2.31 (2
H, m, piperidine CH--), 2.34 (2 H, m, piperidine CH--), 2.92 (4 H,
m, piperidine CH--), 3.32 (2 H, br m, CH.sub.3CH.sub.2N--), 3.55 (2
H, br m, CH.sub.3CH.sub.2N--), 7.13 (2 H, d, J=8.0 Hz, ArH), 7.30
(3 H, m, ArH), 7.38 (1 H, t, J=7.6 Hz, ArH), 7.58 (1 H, s, ArH),
7.66 (1 H, d, J=7.6 Hz, ArH). IR (NaCl) 3307, 2973, 1668, 1615,
1435, 1383, 1289 cm.sup.-1; Anal.Calcd.for
C.sub.24H.sub.29N.sub.3O- .sub.2 2.8 HCl: C, 58.40%; H, 6.49%;
Found: C, 58.46%;H, 6.57%.
[0050]
N,N-Diethyl-4-[1-(2-thiophene)methyl-piperidin-4-ylidene-3-carbamoy-
lphenyl)-methyl]-benzamide (5a).
[0051] To a mixture of
N,N-diethyl-4-[piperidin-4-ylidene(3-carbamoylpheny-
l)-methyl]-benzamide (4, 196 mg, 0.5 mmol),
2-thiophenecarboxaldehyde (112 mg, 1.0 mmol), acetic acid (0.1 mL)
in MeOH (10 mL) was added NaBH.sub.3(CN) (200 mg) in portions. The
reaction mixture was stirred for 4 h at room temperature, and then
quenched with aqueous NH.sub.4Cl, extracted with CH.sub.2Cl.sub.2
(3.times.50 mL). The combined organic phases were washed with
brine, dried over MgSO.sub.4 , and evaporated to give a crude
product, which was purified by flash silica gel column to give the
desired product (5a, 216 mg, 89%). .sup.1H NMR (CDCl.sub.3) .delta.
1.13 (3 H, br m, CH.sub.3CH.sub.2--), 1.22 (3 H, br m,
CH.sub.3CH.sub.2--), 2.36 (2 H, m, piperidine CH--), 2.42 (2 H, m,
piperidine CH--), 2.54 (4 H, m, piperidine CH--), 3.26 (2 H, br m,
CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--), 3.76
(2 H, s, CH.sub.2N--), 5.60 (1 H, br, NH), 6.06 (1 H, br, NH), 6.91
(1 H, s, ArH), 6.94 (1 H, m, ArH), 7.12 (2 H, d, J=8.0 Hz, ArH),
7.26 (1 H, m, ArH), 7.30 (3 H, m, ArH), 7.37 (1 H, t, J=7.6 Hz,
ArH), 7.56 (1 H, s, ArH), 7.64 (1 H, d, J=7.6 Hz, ArH). IR (NaCl)
3352, 2973, 1668, 1614, 1434, 1290 cm.sup.-1; Anal.Calcd.for
C.sub.29H.sub.33N.sub.3O.sub.2S 2.3 HCl: C, 60.95%; H, 6.23%;
Found: C, 60.96 %; H, 6.42%.
[0052]
N,N-Diethyl-4-[1-(2-furfuryl-piperidin-4-ylidene-(3-carbamoylphenyl-
)-methyl]-benzamide (5b).
[0053] Method as for 5a using 4 (196 mg, 0.5 mmol) and
2-furaldehyde (96 mg, 1.0 mmol) provided 5b (158 mg, 67%): .sup.1H
NMR (CDCl.sub.3) .delta. 1.12 (3 H, br m, CH.sub.3CH.sub.2--), 1.22
(3 H, br m, CH.sub.3CH.sub.2--), 2.41 (4 H, m, piperidine CH--),
2.83 (4 H, m, piperidine CH--), 3.27 (2 H, br m,
CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--), 3.88
(2 H, s, CH.sub.2N--), 6.18 (1 H, br, NH), 6.35 (1 H, m, ArH), 6.42
(1 H, m, ArH), 6.86 (1 H, m, NH), 7.12 (2 H, d, J=8.0 Hz, ArH),
7.27 (3 H, m, ArH), 7.32 (1 H, m, ArH), 7.41 (1 H, s, ArH), 7.61 (1
H, s, ArH), 7.69 (1 H, d, J=7.6 Hz, ArH); Anal.Calcd.for
C.sub.29H.sub.33N.sub.3O.sub.3 3.0 HCl: C, 59.95%; H, 6.25%; Found:
C, 59.68%; H, 5.98%.
[0054]
N,N-Diethyl-4-[1-(3-furfuryl-piperidin-4-ylidene-(3-carbamoylphenyl-
)-methyl]-benzamide (5c).
[0055] Method as for 5a using 4 (196 mg, 0.5 mmol) and
3-furaldehyde (96 mg, 1.0 mmol) provided 5c (143 mg, 61%): .sup.1H
NMR (CDCl.sub.3) .delta. 1.13 (3 H, br m, CH.sub.3CH.sub.2--), 1.25
(3 H, br m, CH.sub.3CH.sub.2--), 2.42 (4 H, m, piperidine CH--),
2.70 (4 H, m, piperdine CH--), 3.26 (2 H, br m,
CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--), 3.62
(2 H, s, CH.sub.2N--), 5.80 (1 H, br, NH), 6.42 (1 H, br, NH), 6.46
(1 H, s, ArH), 7.12 (2 H, d, J=8.0 Hz, ArH), 7.28 (3 H, m, ArH),
7.36 (1 H, t, J=7.6 Hz, ArH), 7.42 (2 H, m, ArH), 7.59 (1 H, s,
ArH), 7.66 (1 H, d, J=7.6 Hz, ArH); Anal.Calcd.for
C.sub.29H.sub.33N.sub.3O.sub.3 3.1 HCl: C, 59.58%; H, 6.22%; Found.
C, 59.44%; H, 6.45 %.
[0056]
N,N-Diethyl-4-[1-(2-pyridine)methyl-piperidin-4-ylidene-(3-carbamoy-
lphenyl)-methyl]-benzamide (5d).
[0057] Method as for 5a using 4 (196 mg, 0.5 mmol) and
2-pyridinecarboxaldehyde (107 mg, 1.0 mmol) provided 5d (35 mg,
15%): .sup.1H NMR (CDCl.sub.3) .delta. 1.13 (3 H, br m,
CH.sub.3CH.sub.2--), 1.24 (3 H, br m, CH.sub.3CH.sub.2--),2.38 (2
H, m, piperidine CH--), 2.42 (2 H, m, piperidine CH--), 2.56 (4 H,
m, piperidine CH--), 3.26 (2 H, br m, CH.sub.3CH.sub.2N--), 3.54 (2
H, br m, CH.sub.3CH.sub.2N--), 3.68 (2 H, s, CH.sub.2N--), 5.64 (1
H, br, NH), 6.12 (1 H, br, NH), 7.14 (3 H, m, ArH), 7.31 (3 H, m,
ArH), 7.35 (1 H, m, ArH), 7.38 (1 H, m, ArH), 7.56 (1 H, s, ArH),
7.65 (2 H, m, ArH), 8.58 (1 H, m, ArH).
[0058]
N,N-Diethyl-4-[1-(3-thiophene)methyl-piperidin-4-ylidene-(3-carbamo-
ylphenyl)-methyl]-benzamide (5e).
[0059] Method as for 5a using 4 (196 mg, 0.5 mmol) and
3-thiophenecarboxaldehyde (112 mg, 1.0 mmol) provided 5e (185 mg,
76%): .sup.1H NMR (CDCl.sub.3) .delta. 1.13 (3 H, br m,
CH.sub.3CH.sub.2--), 1.24 (3 H, br m, CHCH.sub.2--), 2.46 (4 H, m,
piperidine CH--), 2.84 (4 H, m, piperidine CH--), 3.26 (2 H, br m,
CH.sub.3CH.sub.2N--), 3.52 (2 H, br m, CH.sub.3CH.sub.2N--), 3.91
(2 H, s, CH.sub.2N--), 5.72 (1 H, br, NH), 6.44 (1 H, br, NH), 7.13
(3 H, m, ArH), 7.26(3 H, m, ArH), 7.36 (3 H, m, ArH), 7.61 (1 H, s,
ArH), 7.68 (1 H, d, J=7.6 Hz, ArH); Anal.Calcd.for
C.sub.29H.sub.33N.sub.3O.sub.2S 2.5 HCl: C, 60.18%; H, 6.18 %;
Found: C, 60.16%; H, 6.49%.
[0060]
N,N-Diethyl-4-[1-(2-thiazole)methyl-piperidin-4-ylidene-(3-carbamoy-
lphenyl)-methyl]-benzamide (5f).
[0061] To a solution of
N,N-diethyl-4-[piperidin-4-ylidene(3-carbamoylphen-
yl)-methyl]-benzamide (4, 300 mg, 0.8 mmol) in 1,2-dichloroethane
(15 mL) was added 2-thiazole carboxaldehyde (94 .mu.L, 1.1 mmol)
and sodium triacetoxyborohydride (228 mg, 1.1 mmol). The reaction
mixture was stirred for 20 h at room temperature, and then quenched
with aqueous NaHCO.sub.3. The aqueous phase was extracted with
CH.sub.2Cl.sub.2 (2.times.20 mL) and the combined organic phases
were dried over MgSO.sub.4, filtered and evaporated. The crude
product was purified by flash chromatography to give product (5f)
as a yellow foam (234 mg, 63% yield).
[0062] The product was dissolved in diciloromethane (5 mL) and a
solution of HCl in ether (1N, 1.4 mL, 3eq.) was added. After 30
minutes the suspension was concentrated and the solid dried .sup.1H
NMR (CDCl.sub.3) .delta. 1.10 (3H, t, J=7 Hz, CH.sub.3) ; 1.21 (3H,
t, J=7 Hz, CH.sub.3); 2.64 (4H, br s, CH.sub.2); 3.27-3.31 (4H, m,
CH.sub.2); 3.48-3.53 (2H, m, CH.sub.2); 3.67 (2H, br s, CH.sub.2);
4.77 (2H, s, NCH.sub.2Ar); 7.27 (2H, d, J=8.5 Hz, Ar--H) ;
7.33-7.37 (3H, m, Ar--H) ; 7.44 (1H, t, J=7.5 Hz, Ar--H); 7.68-7.69
(1H, m, Ar--H); 7.75-7.78 (2H, m, Ar--H); 7.94 (1H, d, J=3.5 Hz,
Ar--H). Anal. Calcd for C.sub.28H.sub.32N.sub.4O.sub.2S.time-
s.2.5HCl: C, 58.00%; H, 6.00%; N, 9.66%; Found: C, 58.00%; H,
5.95%; N, 9.43%.
[0063]
N,N-Diethyl-4-[1-(3-pyridine)methyl-piperidin-4-ylidene-(3-carbamoy-
lphenyl)-methyl]-benzamide (5g).
[0064] Method as for 5f using 4 (176 mg, 0.45 mmol) and
3-pyridinecarboxyaldehyde (60 .mu.L, 0.6 mmol) provided 5g (91.5
mg, 42%): .sup.1H NMR (CDCl.sub.3) .delta. 1.09 (3H, t, J=7 Hz,
CH.sub.3); 1.21 (3H, t, J=7 Hz, CH.sub.3); 2.68-2.75 (4H, m,
CH.sub.2); 3.26-3.28 (4H, m, CH.sub.2); 3.43-3.60 (4H, m,
CH.sub.2); 4.65 (2H, s, NCH.sub.2Ar); 7.27 (2H, d, J=8.5 Hz,
Ar--H); 7.32-7.36 (3H, m, Ar--H); 7.43 (1H, t, J=8 Hz, Ar--H);
7.70-7.71 (1H, m, Ar--H); 7.75-7.78 (1H, m, Ar--H); 8.19 (1H, dd,
J=6, 8 Hz, Ar--H); 8.88 (1H, d, J=8 Hz, Ar--H) ; 8.98 (1H, d, J=6
Hz, Ar--H); 9.21 (1H, s, Ar--H). Anal. Calcd for
C.sub.30H.sub.34N.sub.4O.sub.2.times.3.1HCl.times.0.4H.sub.2O: C,
59.77%; H, 6.34%; N, 9.29%; Found: C, 59.70%; H, 6.36%; N,
9.17%.
[0065]
N,N-Diethyl-4-[1-(2-pyrrole)methyl-piperidin-4-ylidene-(3-carbamoyl-
phenyl)methyl]-benzamide (5h).
[0066] Method as for 5f using 4 (261 mg, 0.7 mmol) and
2-pyrrolecarboxyaldehyde (89 mg, 0.9 mmol) provided 5h (118.5 mg,
38%): .sup.1H NMR (CDCl.sub.3) .delta. 1.09 (3H, t, J=7 Hz,
CH.sub.3); 1.21 (3H, t, J=7 Hz, CH.sub.3) ; 2.46-2.53 (2H, m,
CH.sub.2); 2.65-2.77 (2H, m, CH.sub.2); 2.97-3.04 (2H, m,
CH.sub.2); 3.26-3.30 (2H, m, CH.sub.2); 3.46-3.52 (4H, m,
CH.sub.2); 4.30 (2H, s, NCH.sub.2Ar); 6.33-6.34 (1H, m, Ar--H);
6.85-6.86 (1H, m, Ar--H); 7.24-7.26 (2H, m, Ar--H); 7.30-7.36 (4H,
m, Ar--H); 7.40-7.45 (1H, m, Ar--H); 7.67-7.68 (1H, m, Ar--H);
7.75-7.77 (1H, m, Ar--H). Anal. Calcd for
C.sub.29H.sub.34N.sub.4O.sub.2.- times.1.1HCl.times.1.8H.sub.2O: C,
64.13%; H, 7.18%; N, 10.32%; Found: C, 64.26%; H, 7.15%; N,
9.94%.
[0067]
N,N-Diethyl-4-[1-(4-pyridine)methyl-piperidin-4-ylidene-3-carbamoyl-
phenyl)-methyl]-benzamide (5i).
[0068] Method as for 5f using 4 (329 mg, 0.8 mmol) and
4-pyridinecarboxyaldehyde (112 .mu.L, 1.2 mmol) provided 5i (217
mg, 54%): .sup.1H NMR (CDCl.sub.3) .delta. 1.12 (3H, t, J=7 Hz,
CH.sub.3); 1.24 (3H, t, J=7 Hz, CH.sub.3); 2.67-2.82 (4H, m,
CH.sub.2); 3.22-3.34 (4H, m, CH.sub.2); 3.49-3.65 (4H, m,
CH.sub.2); 4.72 (2H, s, NCH.sub.2Ar); 7.29 (2H, d, J=8.5 Hz,
Ar--H); 7.33-7.40 (3H, m, Ar--H); 7.46 (1H, t, J=8 Hz, Ar--H);
7.72-7.73 (1H, m, Ar--H); 7.78-7.80 (1H, m, Ar--H); 8.37 (2H, d,
J=7 Hz, Ar--H); 9.00 (2H, d, J=7 Hz, Ar--H).
[0069] N,N-Diethyl-4-[1-(4-pyridine)methyl-piperidin-4-ylidene
-(3-carbamoylphenyl)-methyl]-benzarnide (5j).
[0070] Method as for 5f using 4 (313 mg, 0.8 mmol) and
4-imidazolecarboxyaldehyde (108 mg, 1.1mmol) provided 5j (68.2 mg,
18%): .sup.1H NMR (CDCl.sub.3) .delta. 1.12 (3H, t, J=7 Hz,
CH.sub.3) ; 1.23 (3H, t, J=7 Hz, CH.sub.3); 2.63 (2H, t, J=6 Hz,
CH.sub.2) ; 2.68 (2H, t, J=6 Hz, CH.sub.2) ; 3.26-3.36 (6H, m,
CH.sub.2); 3.54 (2H, q, J=7 Hz, CH.sub.2); 4.45 (2H, s,
NCH.sub.2Ar); 7.28 (2H, d, J=8 Hz, Ar--H); 7.32-7.40 (3H, m, Ar--H)
; 7.45 (1H, t, J=8 Hz, Ar--H); 7.66 (1H, s, Ar--H); 7.71 (1H, t,
J=2 Hz, Ar--H); 7.76-7.82 (1H, m, Ar--H); 8.61 (1H, s, Ar--H).
[0071] Pharmaceutical Compositions
[0072] The novel compounds according to the present invention may
be administered orally, intramuscularly, subcutaneously, topically,
intranasally, intraperitoneally, intrathoracially, intravenously,
epidurally, intrathecally, intracerebroventricularly and by
injection into the joints.
[0073] A preferred route of administration is orally, intravenously
or intramuscularly.
[0074] The dosage will depend on the route of administration, the
severity of the disease, age and weight of the patient and other
factors normally considered by the attending physician, when
determining the individual regimen and dosage level as the most
appropriate for a particular patient.
[0075] For preparing pharmaceutical compositions from the compounds
of this invention, inert, pharmaceutically acceptable carriers can
be either solid or liquid. Solid form preparations include powders,
tablets, dispersible granules, capsules, cachets, and
suppositories.
[0076] A solid carrier can be one or more substances which may also
act as diluents, flavoring agents, solubilizers, lubricants,
suspending agents, binders, or tablet disintegrating agents; it can
also be an encapsulating material.
[0077] In powders, the carrier is a finely divided solid which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding properties in suitable proportions and compacted in the
shape and size desired.
[0078] For preparing suppository compositions, a low-melting wax
such as a mixture of fatty acid glycerides and cocoa butter is
first melted and the active ingredient is dispersed therein by, for
example, stirring. The molten homogeneous mixture is then poured
into convenient sized molds and allowed to cool and solidify.
[0079] Suitable carriers are magnesium carbonate, magnesium
stearate, talc, lactose, sugar, pectin, dextrin, starch,
tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a
low-melting wax, cocoa butter, and the like.
[0080] Salts include, but are not limited to, pharmaceutically
acceptable salts. Examples of pharmaceutically acceptable salts
within the scope of the present invention include: acetate,
benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,
calcium acetate, camsylate, carbonate, chloride, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, furnarate,
glucaptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroxynaphthoate, isethionate, lactate, lactobionate, malate,
maleate, mandelate, mesylate, methylbromide, methylnitrate,
methylsulfate, mucate, napsylate, nitrate, pamoate (embonate),
pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,
stearate, subacetate, succinate, sulfate, tannate, tartrate,
teoclate. Examples of pharmaceutically unacceptable salts within
the scope of the present invention include: hydroiodide,
perchlorate, and tetrafluoroborate.
[0081] Preferred pharmaceutically acceptable salts are the
hydrochlorides, sulfates and bitartrates.
[0082] The hydrochloride and sulfate salts are particularly
preferred.
[0083] The term composition is intended to include the formulation
of the active component with encapsulating material as a carrier
providing a capsule in which the active component (with or without
other carriers) is surrounded by a carrier which is thus in
association with it. Similarly, cachets are included. Tablets,
powders, cachets, and capsules can be used as solid dosage forms
suitable for oral administration.
[0084] Liquid from compositions include solutions, suspensions, and
emulsions. Sterile water or water-propylene glycol solutions of the
active compounds may be mentioned as an example of liquid
preparations suitable for parenteral administration. Liquid
compositions can also be formulated in solution in aqueous
polyethylene glycol solution.
[0085] Aqueous solutions for oral administration can be prepared by
dissolving the active component in water and adding suitable
colorants, flavoring agents, stabilizers, and thickening agents as
desired. Aqueous suspensions for oral use can be made by dispersing
the finely divided active component in water together with a
viscous material such as natural synthetic gums, resins, methyl
cellulose, sodium carboxymethyl cellulose, and other suspending
agents known to the pharmaceutical formulation art
[0086] Preferably the pharmaceutical composition is in unit dosage
form. In such form, the composition is divided into unit doses
containing appropriate quantities of the active component The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of the preparations, for example, packeted
tablets, capsules, and powders in vials or ampoules. The unit
dosage form can also be a capsule, cachet, or tablet itself, or it
can be the appropriate number of any of these packaged forms.
[0087] Biological Evaluation
[0088] In Vitro Model
[0089] Cell Culture
[0090] A. Human 293S cells expressing cloned human .mu., .delta.,
and .kappa. receptors and neomycin resistance were grown in
suspension at 37.degree. C. and 5% CO.sub.2 in shaker flasks
containing calcium-free DMEM 10% FBS, 5% BCS, 0.1% Pluronic F-68,
and 600 .mu.g/ml geneticin.
[0091] B. Mouse and rat brains were weighed and rinsed in ice-cold
PBS (containing 2.5 mM EDTA, pH 7.4). The brains were homogenized
with a polytron for 15 sec (mouse) or 30 sec (rat) in ice-cold
lysis buffer (50 mM Tris, pH 7.0, 2.5 mM EDTA, with
phenylmethylsulfonyl fluoride added just prior use to 0.5 MmM from
a 0.5M stock in DMSO:ethanol).
[0092] Membrane Preparation
[0093] Cells were pelleted and resuspended in lysis buffer (50 mM
Tris, pH 7.0, 2.5 mM EDTA, with PMSF added just prior to use to 0.1
mM from a 0.1 M stock in ethanol), incubated on ice for 15 min,
then homogenized with a polytron for 30 sec. The suspension was
spun at 1000 g (max) for 10 min at 4.degree. C. The supermatant was
saved on ice and the pellets resuspended and spun as before. The
supernatants from both spins were combined and spun at 46,000 g
(max) for 30 min. The pellets were resuspended in cold Tris buffer
(50 mM Tris/Cl, pH 7.0) and spun again. The final pellets were
resuspended in membrane buffer (50 mM Tris, 0.32 M sucrose, pH
7.0). Aliquots (1 ml) in polypropylene tubes were frozen in dry
ice/ethanol and stored at -70.degree. C. until use. The protein
concentrations were determined by a modified Lowry assay with
sodium dodecyl sulfate.
[0094] Binding Assays
[0095] Membranes were thawed at 37.degree. C., cooled on ice,
passed 3 times through a 25-gauge needle, and diluted into binding
buffer (50 mM Tris, 3 mM MgCl.sub.2, 1 mg/ml BSA (Sigma A-7888), pH
7.4, which was stored at 4.degree. C. after filtration through a
0.22 m filter, and to which had been freshly added 5 .mu.g/ml
aprotinin, 10 .mu.M bestatin, 10.mu.M diprotin A, no DTT). Aliquots
of 100 .mu.l were added to iced 12.times.75 mm polypropylene tubes
containing 100 .mu.l of the appropriate radioligand and 100 .mu.l
of test compound at various concentrations. Total (TB) and
nonspecific (NS) binding were determined in the absence and
presence of 10 .mu.M naloxone respectively. The tubes were vortexed
and incubated at 25.degree. C. for 60-75 min, after which time the
contents are rapidly vacuum-filtered and washed with about 12
ml/tube iced wash buffer (50 mM Tris, pH 7.0, 3 mM MgCl.sub.2)
through GF/B filters (Whatman) presoaked for at least 2 h in 0.1%
polyethyleneimine. The radioactivity (dpm) retained on the filters
was measured with a beta counter after soaking the filters for at
least 12 h in minivials containing 6-7 ml scintillation fluid. If
the assay is set up in 96-place deep well plates, the filtration is
over 96-place PEI-soaked unifilters, which were washed with
3.times.1 ml wash buffer, and dried in an oven at 55.degree. C. for
2 h. The filter plates were counted in a TopCount (Packard) after
adding 50 .mu.l MS-20 scintillation fluid/well.
[0096] Functional Assays
[0097] The agonist activity of the compounds is measured by
determining the degree to which the compounds receptor complex
activates the binding of GTP to G-proteins to which the receptors
are coupled. In the GTP binding assay, GTP[.gamma.].sup.35S is
combined with test compounds and membranes from HEK-293S cells
expressing the cloned human opioid receptors or from homogenised
rat and mouse brain. Agonists stimulate GTP[.gamma.].sup.35S
binding in these membranes. The EC.sub.50 and E.sub.max values of
compounds are determined from dose-response curves. Right shifts of
the dose response curve by the delta antagonist naltrindole are
performed to verify that agonist activity is mediated through delta
receptors.
[0098] Data Analysis
[0099] The specific binding (SB) was calculated as TB-NS, and the
SB in the presence of various test compounds was expressed as
percentage of control SB. Values of IC.sub.50 and Hill coefficient
(n.sub.H) for ligands in displacing specifically bound radioligand
were calculated from logit plots or curve fitting programs such as
Ligand, GraphPad Prism, SigmaPlot, or ReceptorFit. Values of
K.sub.i were calculated from the Cheng-Prussoff equation.
Mean.+-.S.E.M. values of IC.sub.50, K.sub.i and n.sub.H were
reported for ligands tested in at least three displacement curves.
Biological data are tabulated on the following pages in Table
1.
1TABLE 1 Biological data. MOLECULAR HDELTA (nM) RAT BRAIN MOUSE
BRAIN Ex. # STRUCTURE IC.sub.50 EC.sub.50 % EMax EC.sub.50 % EMax
EC.sub.50 % EMax 3b 16 0.548 0.091 93.775 0.403 178.94 0.539 164.82
5a 17 0.373 0.158 98.107 0.613 181.01 0.818 170.24 5b 18 0.282
0.633 149.28 5c 19 0.246 0.367 170.4 5d 20 0.278 0.396 179.89 5e 21
0.235
[0100] Receptor Saturation Experiments
[0101] Radioligand K.sub.67 values were determined by performing
the binding assays on cell membranes with the appropriate
radioligands at concentrations ranging from 0.2 to 5 times the
estimated K.sub.67 (up to 10 times if amounts of radioligand
required are feasible). The specific radioligand binding was
expressed as pmole/mg membrane protein. Values of K.sub.67 and
B.sub.max from individual experiments were obtained from nonlinear
fits of specifically bound (B) vs. nM free (F) radioligand from
individual according to a one-site model.
[0102] Determination of Mechano-Allodynia Using Von Frey
Testing
[0103] Testing was performed between 08:00 and 16:00 h using the
method described by Chaplan et al. (1994). Rats were placed in
Plexiglas cages on top of a wire mesh bottom which allowed access
to the paw, and were left to habituate for 10-15 min. The area
tested was the mid-plantar left hind paw, avoiding the less
sensitive foot pads. The paw was touched with a series of 8 Von
Frey hairs with logarithmically incremental stiffness (0.41, 0.69,
1.20, 2.04, 3.63, 5.50, 8.51, and 15.14 grams; Stoelting, III,
USA). The von Frey hair was applied from underneath the mesh floor
perpendicular to the plantar surface with sufficient force to cause
a slight buckling against the paw, and held for approximately 6-8
seconds. A positive response was noted if the paw was sharply
withdrawn. Flinching immediately upon removal of the hair was also
considered a positive response. Ambulation was considered an
ambiguous response, and in such cases the stimulus was
repeated.
[0104] Testing Protocol
[0105] The animals were tested on postoperative day 1 for the
FCA-treated group. The 50% withdrawal threshold was determined
using the up-down method of Dixon (1980). Testing was started with
the 2.04 g hair, in the middle of the series. Stimuli were always
presented in a consecutive way, whether ascending or descending. In
the absence of a paw withdrawal response to the initially selected
hair, a stronger stimulus was presented; in the event of paw
withdrawal, the next weaker stimulus was chosen. Optimal threshold
calculation by this method requires 6 responses in the immediate
vicinity of the 50% threshold, and counting of these 6 responses
began when the first change in response occurred, e.g. the
threshold was first crossed. In cases where thresholds fell outside
the range of stimuli, values of 15.14 (normal sensitivity) or 0.41
(maximally allodynic) were respectively assigned. The resulting
pattern of positive and negative responses was tabulated using the
convention, X=no withdrawal; O=withdrawal, and the 50% withdrawal
threshold was interpolated using the formula:
50% g threshold=10.sup.(Xf+k.delta.)/10,000
[0106] where Xf=value of the last von Frey hair used (log units);
k=tabular value (from Chaplan et al. (1994)) for the pattern of
positive /negative responses; and .delta.=mean difference between
stimuli (log units). Here .delta.=0.224.
[0107] Von Frey thresholds were converted to percent of maximum
possible effect (% MPE), according to Chaplan et al. 1994. The
following equation was used to compute % MPE: 1 % MPE = Drug
treated threshold ( g ) - allodynia threshold ( g ) .times. 100
Control threshold ( g ) - allodynia threshold ( g )
[0108] Administration of Test Substance
[0109] Rats were injected (subcutaneously, intraperitoneally,
intravenously or orally) with a test substance prior to von Frey
testing, the time between administration of test compound and the
von Frey test varied depending upon the nature of the test
compound
[0110] Writhing Test
[0111] Acetic acid will bring abdominal contractions when
administered intraperitoneally in mice. These will then extend
their body in a typical pattern. When analgesic drugs are
administered, this described movement is less frequently observed
and the drug selected as a potential good candidate.
[0112] A complete and typical Writhing reflex is considered only
when the following elements are present: the animal is not in
movement; the lower back is slightly depressed; the plantar aspect
of both paws is observable. In this assay, compounds of the present
invention demonstrate significant inhibition of writhing responses
after oral dosing of 1-100 .mu.mol/kg.
[0113] (i) Solutions Preparation
[0114] Acetic acid (AcOH): 120 .mu.L of Acetic Acid is added to
19.88 ml of distilled water in order to obtain a final volume of 20
ml with a final concentration of 0.6% AcOH. The solution is then
mixed (vortex) and ready for injection.
[0115] Compound (drug): Each compound is prepared and dissolved in
the most suitable vehicle according to standard procedures.
[0116] (ii) Solutions Administration
[0117] The compound (drug) is administered orally,
intraperitoneally (i.p.), subcutaneously (s.c.) or intravenously
(i.v.)) at 10 ml/kg (considering the average mice body weight) 20,
30 or 40 minutes (according to the class of compound and its
characteristics) prior to testing. When the compound is delivered
centrally: Intraventricularly (i.c.v.) or intrathecally (i.t.) a
volume of 5 .mu.L is administered.
[0118] The AcOH is administered intraperitoneally (i.p.) in two
sites at 10 ml/kg (considering the average mice body weight)
immediately prior to testing.
[0119] (iii) Testing
[0120] The animal (mouse) is observed for a period of 20 minutes
and the number of occasions (Writhing reflex) noted and compiled at
the end of the experiment Mice are kept in individual "shoe box"
cages with contact bedding. A total of 4 mice are usually observed
at the same time: one control and three doses of drug.
* * * * *